Fluorescent assays are now commonly used for diagnostic and research purposes to detect and measure the quantity of a wide variety of immunological and non-immunological substances. In fluorescent immunoassays, the test sample may be prepared for fluorometric measurement in a variety of different ways.
For example, a fluorescently labeled reactant may be immunologically reacted with the immunological substance of interest which is usually directly or indirectly immobilized on a solid phase. Following separation of the nonspecific substances which are not attached to the substance of interest, the fluorescence of the reaction product is measured to determine the amount of the substance of interest. In another type of fluorescent immunoassay, an enzyme labeled reactant is attached to the immunological substance of interest, and a fluorogenic substrate is catalyzed by the enzyme label to yield a fluorescent product which can be fluorescently measured to determine the quantity of the substance of interest.
In carrying out fluorescent assays of the foregoing type and also other types, microtest plates (or microtitration plates, as they are also called) and strips of microtest wells are often used. Microtest plates are formed with a multiplicity of wells which are joined together in a molded one-piece structure for containing microliter quantities of fluid samples. Examples of a microtest plate and microtest wells in strip form are described in U.S. Pat. No. 4,154,795 which issued to A. C. Thorne on May 15, 1979.
The use of microtest plates and microtest strips of wells in fluorescent and other types of assays offers several important advantages. First, they permit the mass preparation of a large number of test sample solutions at the same time. Second, they are more convenient to handle as compared with individual test tubes. Third, they can easily and inexpensively be washed. Fourth, they are inexpensive and disposable. Fifth, they are customarily formed from plastic materials which are not fragile like glass. Sixth, they can be made from a material having an ability to attract certain molecules such as protein molecules so that they can serve as a solid phase in an immunoassay. Polystyrene and polyvinyl chloride are commonly used for this purpose and exhibit acceptable protein binding properties for attracting protein molecules.
The use of plastic materials permits the microtest plates and strips to be manufactured by low cost, mass production molding techniques.
For fluorescent assays, however, molded microtest plates and strips of the type described above have a serious drawback in that the moldable materials customarily used for low cost manufacture exhibit a substantial level of native fluorescence, particularly at the exciting light wave lengths commonly employed in fluorometers. When used to hold a test sample in a fluorometer for making a fluorometric measurement they therefore will unavoidably be excited along with the test sample by the fluorometer's exciting light. As a result, the microtest plates, strips or their individual wells will fluoresce to produce spurious light emissions which interfere with and impair an accurate measurement of the intensity of the light emitted from the excited test sample itself. These spurious light emissions have the objectionable effect of creating a noise signal in the fluorometer's detector to significantly reduce the signal-to-noise ratio.
One solution to the foregoing problem is to equip the fluorometers with special, sample-holding vessels made of non-fluorescent or low-fluorescing materials such as quartz or certain kinds of glass. In addition, optical grade Teflon has been suggested as a non-fluorescing material for making fluorometer flow cells in U.S. Pat. No. 4,008,397 which issued to J. J. Zdrodowski on Feb. 15, 1977. Also, an Italian company called Kartell is marketing a molded cuvette of undisclosed plastic material which is claimed to have a sufficiently low level of fluorescence to make it suitable for holding samples in a fluorometer. Although the level of fluorescence of the Kartell cuvette is lower than that of clear polystyrene, it nevertheless is significant.
The foregoing cuvettes and test tubes share a common drawback in that they are each capable of holding only a single test sample. Vessels of this type are therefore less convenient to work with as compared with microtest plates or strips. More particularly, they are not suitable for the mass preparation of test samples as with microtest plates and strips. In addition, they are more difficult and more expensive to wash in the course of performing the various steps in an immunoassay.
Furthermore, quartz and glass are not suitable for making the intricately shaped microtest plates and strips because the manufacturing costs would be prohibitively high. In addition, the protein binding properties of quartz and glass are inferior to the protein binding properties of polystyrene and other plastics which are customarily used for making microtest plates and strips. They therefore are not as suitable as polystyrene and other plastics for defining a solid phase in an immunoassay. Finally, they are fragile and are not intended to be disposable.
Because of the disadvantages associated with cuvettes or tubes, it is often desirable and sometimes necessary to prepare the test samples in a standard microtest plate or strip and then to transfer the samples to the cuvettes or tubes for measurement. Transferring the test samples is time consuming, inconvenient and increases the cost of the fluoroescent assay.
From the foregoing, it is clear that, on the one hand, there are easily moldable materials (such as polystyrene and polyvinyl chloride) which are suitable for use in low cost, mass production of microtest plates and other sample-holding vessels, but which exhibit a substantial level of native fluorescence to create a problem in measuring the fluorescence of test samples. On the other hand, there are materials such as quartz and certain kinds of glass which have low levels of native fluorescence to avoid the foregoing problem, but which are unsuitable for the low cost production of microtest plates and strips.